Methods and compositions for generating a mouse

Abstract

Methods of generating modified embryos and mammals by introduction of donor cells into an early stage embryo are provided, such that the resulting embryo and animal generated therefrom has a significant contribution to all tissues from the donor cells and is capable of transmitting the donor cell DNA.

We claim: 
     
       1. A method for generating a mouse embryo, comprising:
 (a) introducing mouse donor cells into an 8-cell diploid host mouse embryo, wherein the donor cells comprise embryonic stem (ES) cells from an inbred mouse strain; 
 (b) culturing the 8-cell host embryo of (a) to the blastocyst stage; 
 wherein at least 90% of the cells of a mouse that develops from the blastocyst are derived from the donor cells. 
 
     
     
       2. The method of  claim 1 , wherein the inbred mouse strain is selected from the group consisting of 129, C57BL/6, or BalbC. 
     
     
       3. The method of  claim 1 , wherein the host embryo is an inbred strain. 
     
     
       4. The method of  claim 3 , wherein the inbred strain is C57BL/6. 
     
     
       5. The method of  claim 1 , wherein the host embryo comprises an outbred strain. 
     
     
       6. The method of  claim 5 , wherein the outbred strain is Swiss Webster. 
     
     
       7. The method of  claim 1 , wherein the culturing in step (b) is conditioned by a growth factor. 
     
     
       8. The method of  claim 7 , wherein the growth factor is a protein of the Wnt family. 
     
     
       9. The method of  claim 8 , wherein the protein of the Wnt family is Wnt3a. 
     
     
       10. The method of  claim 1 , wherein the 8-cell diploid host mouse embryo comprises a zona pellucida, and wherein the mouse donor cells are introduced into the host mouse embryo through an opening in the zona pellucida. 
     
     
       11. The method of  claim 10 , wherein the opening is created by a laser. 
     
     
       12. The method of  claim 1 , wherein the mouse donor cells are homozygous for a genetic modification. 
     
     
       13. The method of  claim 1 , further comprising introducing the embryo of (b) into a surrogate mouse mother for gestation. 
     
     
       14. The method of  claim 1 , wherein the mouse donor cells are heterozygous for a genetic modification. 
     
     
       15. The method of  claim 1 , wherein (i) at least 95% of the cells of a mouse that develops from the blastocyst are derived from the donor cells, or (ii) at least 99% of the cells of a mouse that develops from the blastocyst are derived from the donor cells. 
     
     
       16. The method of  claim 12 , wherein the mouse donor cells homozygous for the genetic modification are generated by a process selected from the group consisting of gene conversion, targeting both alleles of the same gene, and targeting either an X- or a Y-linked gene in the ES cells. 
     
     
       17. The method of  claim 12 , further comprising gestating the genetically modified mouse embryo to generate a genetically modified mouse, wherein at least 90%, at least 95%, or at least 99% of the cells in the genetically modified mouse are homozygous for the genetic modification. 
     
     
       18. The method of  claim 17 , wherein at least 90% of the germ cells in the genetically modified mouse are homozygous for the genetic modification. 
     
     
       19. The method of  claim 14 , further comprising gestating the genetically modified mouse embryo to generate a genetically modified mouse, wherein at least 90%, at least 95%, or at least 99% of the cells in the genetically modified mouse are heterozygous for the genetic modification. 
     
     
       20. The method of  claim 19 , wherein at least 90% of the germ cells in the genetically modified mouse are heterozygous for the genetic modification. 
     
     
       21. The method of  claim 17 , wherein the donor cells are female XO cells and the genetically modified mouse is an XO mouse. 
     
     
       22. The method of  claim 19 , wherein the donor cells are female XO cells and the genetically modified mouse is an XO mouse. 
     
     
       23. The method of  claim 1 , wherein the donor cells are female XO cells. 
     
     
       24. The method of  claim 12 , wherein the genetically modified donor cells are generated by:
 (a) obtaining a large cloned genomic fragment containing a DNA sequence of interest; 
 (b) genetically modifying the large cloned genomic fragment of (a) to create a large targeting vector for use in the donor cells (LTVEC); 
 (c) introducing the LTVEC of (b) into the donor cells to modify an endogenous gene or a chromosomal locus in the cells; and 
 (d) detecting the donor cells that are genetically modified to comprise the DNA sequence of interest. 
 
     
     
       25. The method of  claim 14 , wherein the genetically modified donor cells are generated by:
 (a) obtaining a large cloned genomic fragment containing a DNA sequence of interest; 
 (b) genetically modifying the large cloned genomic fragment of (a) to create a large targeting vector for use in the donor cells (LTVEC); 
 (c) introducing the LTVEC of (b) into the donor cells to modify an endogenous gene or a chromosomal locus in the cells; and 
 (d) detecting the donor cells that are genetically modified to comprise the DNA sequence of interest. 
 
     
     
       26. An 8-cell diploid mouse embryo comprising mouse donor cells introduced under the zona pellucida, wherein the donor cells comprise ES cells from an inbred mouse strain and are heterozygous or homozygous for a genetic modification, wherein at least 90% of the cells of a mouse that develops from the embryo are derived from the donor cells. 
     
     
       27. The 8-cell diploid mouse embryo of  claim 26 , wherein the donor ES cells are from an inbred mouse strain selected from the group consisting of 129, C57BL/6, and BalbC.